This invention relates to methods for using fusion proteins produced in microbial hosts and more particularly, relates to assays utilizing CKS fusion proteins.
It is well established that prokaryotic or eukaryotic proteins can be expressed in microbial hosts where such proteins are not normally present in such hosts (i.e. are "heterologous" to the cells). Generally, such protein expression is accomplished by inserting the DNA sequence which codes for the protein of interest downstream from a control region (e.g. a lac operon) in plasmid DNA, which plasmid is inserted into the cell to "transform" the cell so it can produce (or "express") the protein of interest.
Despite this conceptually straightforward procedure, there are a number of obstacles in getting a cell to synthesize a heterologous protein and subsequently, to detect and recover the protein. The heterologous gene may not be efficiently transcribed into messenger RNA (mRNA). The mRNA may be unstable and degrade prior to translation into the protein. The ribosome binding site (RBS) present on the mRNA may only poorly initiate translation. The heterologous protein produced may be unstable in the cell or it may be toxic to the cell. If no antibodies to the protein are available or if there is no other way to assay for the protein, it may be difficult to detect the synthesized protein. Lastly, even if the protein is produced, it may be difficult to purify.
Fusion systems provide a means of solving many of the aforementioned problems. The "carrier" portion of the hybrid gene, typically found on the 5' end of the gene, provides the regulatory regions for transcription and translation as well as providing the genetic code for a peptide which facilitates detection (Shuman et al., J. Biol. Chem. 255:168 [1980]) and/or purification (Moks et al., Bio/Technology 5:379 [1987]). Frequently, potential proteolytic cleavage sites are engineered into the fusion protein to allow for the removal of the homologous peptide portion (de Geus et al., Nucleic Acids Res. 15:3743 [1987]; Nambiar et al., Eur. J. Biochem. 163:67 [1987]; Imai et al., J. Biochem. 100:425 [1986]).
When selecting a carrier gene for a fusion system, in addition to detectability and ease of purification, it would be extremely advantageous to start with a highly expressed gene. Expression is the result of not only efficient transcription and translation but also protein stability and benignity (the protein must not harm or inhibit the cell host). Such expression is advantageous because it can enable the production of such fusion proteins for use in assays.
It is known to use cell lysates from microorganisms such as viruses, bacteria, and the like. However, it oftentimes is difficult to produce viral lysates at such levels of purity as is required by current assays. Also, the production of viral or bacterial lysates involves exposing individuals to the microorganism in order to obtain the lysate. Commercial assays are being developed which use fusion proteins of such microorganisms instead of viral lysates as assay reagents.
In addition to their use in assays to detect the presence of infectious agents, it has been discovered that fusion proteins can be useful to detect immunosuppressive agents. For example, in pending U.S. patent application Ser. No. 07/893,858 is described the use of CKS fusion proteins in assays to detect the presence and/or amount of immunosuppressive agents such as FK-506, cyclosporin and rapamycin which are macrocyclic drugs of Streptomycete origin having in vivo and in vitro immunosuppressive properties (A. W. Thomson, Immunol. Today 10:6-9 [1989] and B. D. Kahan et al., Transp. 52:185-191 [1991]), which enjoys common ownership and is incorporated herein by reference.